Novel esterases and uses thereof

ABSTRACT

The present invention relates to novel esterase, more particularly to esterase variants having improved thermostability compared to the esterase of SEQ ID N o  1 and the uses thereof for degrading polyester containing material, such as plastic products. The esterases of the invention are particularly suited to degrade polyethylene terephthalate, and material containing polyethylene terephthalate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/812,405, filed Mar. 9, 2020, now U.S. Pat. No. 11,414,651, which is acontinuation of U.S. application Ser. No. 16/317,160, filed Jan. 11,2019, now U.S. Pat. No. 10,584,320, which is the U.S. national stageapplication of International Patent Application No. PCT/EP2017/067582,filed Jul. 12, 2017.

The Sequence Listing for this application is labeled “Seq-List.xml”which was created on Jul. 10, 2022 and is 2559 bytes. The entire contentof the sequence listing is incorporated herein by reference in itsentirety.

The present invention relates to novel esterases, more particularly toesterases having improved thermostability compared to a parent esteraseand the uses thereof for degrading polyester containing material, suchas plastic products. The esterases of the invention are particularlysuited to degrade polyethylene terephthalate, and material containingpolyethylene terephthalate.

BACKGROUND

Esterases are able to catalyze the hydrolysis of a variety of polymers,including polyesters. In this context, esterases have shown promisingeffects in a number of industrial applications, including as detergentsfor dishwashing and laundry applications, as degrading enzymes forprocessing biomass and food, as biocatalysts in detoxification ofenvironmental pollutants or for the treatment of polyester fabrics inthe textile industry. In the same way, the use of esterases as degradingenzymes for hydrolyzing polyethylene terephthalate (PET) is ofparticular interest. Indeed, PET is used in a large number of technicalfields, such as in the manufacture of clothes, carpets, or in the formof a thermoset resin for the manufacture of packaging or automobileplastics or other parts, and PET accumulation in landfills becomes anincreasing ecological problem.

Among esterases, cutinases, also known as cutin hydrolases (EC3.1.1.74), are of particular interest. Cutinases have been identifiedfrom various fungi (P. E. Kolattukudy in “Lipases”, Ed. B. Borg-strómand H. L. Brockman, Elsevier 1984, 471-504), bacteria and plant pollen.Recently, metagenomics approaches have led to identification ofadditional esterases.

The enzymatic degradation is considered as an interesting solution todecrease such plastic waste accumulation. Indeed, enzymes may acceleratehydrolysis of polyester containing material, and more particularly ofplastic products, even up to the monomer level. Furthermore, thehydrolysate (i.e., monomers and oligomers) can be recycled as materialfor synthesizing new polymers.

In this context, several esterases have been identified as candidatedegrading enzymes. For instance, several variants of the esterase(cutinase) of Fusarium solani pisi have been published (Appl. Environm.Microbiol. 64, 2794-2799, 1998; Proteins: Structure, Function andGenetics 26,442-458,1996).

However, most of these esterases are not efficient at an industriallevel, because of their poor resistance to high temperatures.Accordingly, there is still a need for esterases with improvedthermostability that may be used for degrading polyester at anindustrial level and with high yield.

SUMMARY OF THE INVENTION

The present invention provides new variants of esterase exhibitingincreased thermostability compared to a parent, or wild-type esterase.These esterases are particularly useful in processes for degradingplastic material and product, such as plastic material and productcontaining PET. More particularly, the present invention providesvariants of an esterase having the amino acid sequence as set forth inSEQ ID N^(o) 1, that corresponds to the amino acids 36 to 293 of theamino acid sequence of the metagenome-derived cutinase described inSulaiman et al., Appl Environ Microbiol. 2012 March, or to the aminoacids 36 to 293 of the amino acid sequence referenced G9BY57 inSwissProt.

In this regard, it is an object of the invention to provide an esterasewhich (i) has at least 75%, 80%, 85%, 90%, 95% or 99% identity to thefull length amino acid sequence set forth in SEQ ID N^(o) 1, (ii)contains at least one amino acid modification as compared to SEQ IDN^(o) 1, (iii) has a polyester degrading activity and (iv) exhibitsincreased thermostability as compared to the esterase of SEQ ID N^(o) 1.

More particularly, the esterase of the invention comprises one or moreamino acid modification(s) as compared to SEQ ID N^(o) 1 at position(s)selected from D203+S248, E173, L202, N204, F208, A172+A209, G39, A103,L82, G53, L104, L107, L119, A121, L124, I54, M56, L70, L74, A127, V150,L152, L168, V170, P196, V198, V200, V219, Y220, T221, S223, W224, M225,L239, T252, N253, H256, S1, Y4, Q5, R6, N9, S13, T16, S22, T25, Y26,S34, Y43, S48, T50, R72, S98, N105, R108, S113, N122, S145, K147, T160,N162, S181, Q189, N190, S193, T194, N204, S212, N213, N231, T233, R236,Q237, N241, N243, N254, R255 and Q258 wherein the positions are numberedby reference to the amino acid sequence set forth in SEQ ID N^(o) 1.

In a particular embodiment, the variant esterase of the inventioncomprises one or more amino acid substitution(s) as compared to SEQ IDN^(o) 1 at position(s) selected from D203+S248, E173, N204, L202, F208,and V170. Preferably, the variant esterase of the invention comprises atleast amino acid substitution(s) as compared to SEQ ID N^(o) 1 atposition(s) selected from D203+S248 and F208.

In another particular embodiment, the variant esterase of the inventioncomprises one or more amino acid substitution(s) as compared to SEQ IDN^(o) 1 at a position selected from T61, Y92 and V177, wherein thepositions are numbered by reference to the amino acid sequence set forthin SEQ ID N^(o) 1, and wherein the substitutions are different fromT61A/G, Y92A and V177A. Preferably, the variant esterase of theinvention comprises one or more amino acid substitution(s) as comparedto SEQ ID N^(o) 1, selected from V1771, Y92G, Y92P, Y92P+F208W and T61M.

In a particular embodiment, the variant esterases of the invention maycomprise, as compared to the esterase of SEQ ID N^(o) 1:

-   -   at least one additional disulphide bridge; and/or    -   at least one additional salt bridge; and/or    -   at least one mutation of an amino acid residue located in a void        solvent-excluded cavity of the esterase; and/or    -   a suppression of at least one N- and/or C-terminal amino acid        residue.

It is another object of the invention to provide a nucleic acid encodingan esterase of the invention. The present invention also relates to anexpression cassette or an expression vector comprising said nucleicacid, and to a host cell comprising said nucleic acid, expressioncassette or vector.

It is a further object of the invention to provide a method of producingan esterase comprising:

(a) culturing the host cell according to the invention under suitableconditions to express the nucleic acid encoding the esterase; andoptionally

(b) recovering said esterase from the cell culture.

The present invention also relates to a method of degrading a plasticproduct containing at least one polyester comprising

(a) contacting the plastic product with an esterase or the host cellaccording to the invention, thereby degrading the plastic product; andoptionally

(b) recovering monomers and/or oligomers.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The present disclosure will be best understood by reference to thefollowing definitions.

Herein, the terms “peptide”, “polypeptide”, “protein”, “enzyme”, referto a chain of amino acids linked by peptide bonds, regardless of thenumber of amino acids forming said chain. The amino acids are hereinrepresented by their one-letter or three-letters code according to thefollowing nomenclature: A: alanine (Ala); C: cysteine (Cys); D: asparticacid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine(Gly); H: histidine (His); I: isoleucine (Ile); K: lysine (Lys); L:leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline(Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T:threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y: tyrosine(Tyr).

The term “esterase” refers to an enzyme which belongs to a class ofhydrolases classified as EC 3.1.1 according to Enzyme Nomenclature thatcatalyzes the hydrolysis of esters into an acid and an alcohol. The term“cutinase” or “cutin hydrolase” refers to an esterase classified as EC3.1.1.74 according to Enzyme Nomenclature that is able to catalyse thechemical reaction of production of cutin monomers from cutin and water.

The terms “wild-type protein” or “parent protein” are usedinterchangeably and refer to the non-mutated version of a polypeptide asit appears naturally. In the present case, the parent esterase refers tothe esterase having the amino acid sequence as set forth in SEQ ID N^(o)1.

Accordingly, the terms “mutant” and “variant” may be usedinterchangeably to refer to polypeptides derived from SEQ ID N^(o) 1 andcomprising a modification or an alteration, i.e., a substitution,insertion, and/or deletion, at one or more (e.g., several) positions andhaving a polyester degrading activity. The variants may be obtained byvarious techniques well known in the art. In particular, examples oftechniques for altering the DNA sequence encoding the wild-type protein,include, but are not limited to, site-directed mutagenesis, randommutagenesis and synthetic oligonucleotide construction.

The term “modification” or “alteration” as used herein in relation to aposition or amino acid means that the amino acid in the particularposition has been modified compared to the amino acid of the wild-typeprotein.

A “substitution” means that an amino acid residue is replaced by anotheramino acid residue. Preferably, the term “substitution” refers to thereplacement of an amino acid residue by another selected from thenaturally-occurring standard 20 amino acid residues, rare naturallyoccurring amino acid residues (e.g. hydroxyproline, hydroxylysine,allohydroxylysine, 6-N-methylysine, N-ethylglycine, N-methylglycine,N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline,pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), andnon-naturally occurring amino acid residue, often made synthetically,(e.g. cyclohexyl-alanine). Preferably, the term “substitution” refers tothe replacement of an amino acid residue by another selected from thenaturally-occurring standard 20 amino acid residues (G, P, A, V, L, I,M, C, F, Y, W, H, K, R, Q, N, E, D, S and T). The sign “+” indicates acombination of substitutions. In the present document, the followingterminology is used to designate a substitution: L82A denotes that aminoacid residue (Leucine, L) at position 82 of the parent sequence ischanged to an Alanine (A). A121V/I/M denotes that amino acid residue(Alanine, A) at position 121 of the parent sequence is substituted byone of the following amino acids: Valine (V), Isoleucine (I), orMethionine (M). The substitution can be a conservative ornon-conservative substitution. Examples of conservative substitutionsare within the groups of basic amino acids (arginine, lysine andhistidine), acidic amino acids (glutamic acid and aspartic acid), polaramino acids (glutamine, asparagine and threonine), hydrophobic aminoacids (methionine, leucine, isoleucine, cysteine and valine), aromaticamino acids (phenylalanine, tryptophan and tyrosine), and small aminoacids (glycine, alanine and serine).

The term “deletion”, used in relation to an amino acid, means that theamino acid has been removed or is absent.

The term “insertion” means that one or more amino acids have been added.

Unless otherwise specified, the positions disclosed in the presentapplication are numbered by reference to the amino acid sequence setforth in SEQ ID N^(o) 1.

As used herein, the term “sequence identity” or “identity” refers to thenumber (or fraction expressed as a percentage %) of matches (identicalamino acid residues) between two polypeptide sequences. The sequenceidentity is determined by comparing the sequences when aligned so as tomaximize overlap and identity while minimizing sequence gaps. Inparticular, sequence identity may be determined using any of a number ofmathematical global or local alignment algorithms, depending on thelength of the two sequences. Sequences of similar lengths are preferablyaligned using a global alignment algorithms (e.g. Needleman and Wunschalgorithm; Needleman and Wunsch, 1970) which aligns the sequencesoptimally over the entire length, while sequences of substantiallydifferent lengths are preferably aligned using a local alignmentalgorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981)or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)).Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer softwareavailable on internet web sites such as blast.ncbi.nlm.nih.gov/orebi.ac.uk/Tools/emboss/). Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, % amino acid sequenceidentity values refers to values generated using the pair wise sequencealignment program EMBOSS Needle that creates an optimal global alignmentof two sequences using the Needleman-Wunsch algorithm, wherein allsearch parameters are set to default values, i.e. Scoringmatrix=BLOSUM62, Gap open=10, Gap extend=0.5, End gap penalty=false, Endgap open=10 and End gap extend=0.5.

The terms “disulphide bridge”, “disulphide bond” and “S—S bond” are usedinterchangeably and refer to a covalent bond between sulphur atoms oftwo cysteines.

The term “salt bridge”, or “ion-pair”, refers to a noncovalentelectrostatic interaction between two residues with opposite charges ina protein. A salt bridge most often is formed between the anioniccarboxylate (RCOO⁻) of either aspartic acid or glutamic acid and thecationic ammonium (RNH₃ ⁺) of lysine or the guanidinium (RNHC(NH₂)₂ ⁺)moiety of arginine. Other amino acid residues with ionizable sidechains, such as histidine, tyrosine, serine, threonine and cysteine canalso be part of a salt bridge.

The term “glycosylated”, in relation to a polypeptide, means that one orseveral glycans are attached to at least one amino acid residue of thepolypeptide. In the context of the invention, glycosylation encompassesN-linked glycans, attached to the amide nitrogen of asparagine residue,O-linked glycans attached to the hydroxyl oxygen of serine or tyrosineresidues, C-linked glycans attached to a carbon of a tryptophan residue.

The “protein conformation” or “crystal structure” refers to the threedimensional structure of the protein.

The term “recombinant” refers to a nucleic acid construct, a vector, apolypeptide or a cell produced by genetic engineering.

The term “expression”, as used herein, refers to any step involved inthe production of a polypeptide including, but being not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

The term “expression cassette” denotes a nucleic acid constructcomprising a coding region, i.e. a nucleic acid of the invention, and aregulatory region, i.e. comprising one or more control sequences,operably linked.

As used herein, the term “expression vector” means a DNA or RNA moleculethat comprises an expression cassette of the invention. Preferably, theexpression vector is a linear or circular double stranded DNA molecule.

A “polymer” refers to a chemical compound or mixture of compounds whosestructure is constituted of multiple monomers (repeat units) linked bycovalent chemical bonds. Within the context of the invention, the termpolymer includes natural or synthetic polymers, constituted of a singletype of repeat unit (i.e., homopolymers) or of a mixture of differentrepeat units (i.e., copolymers or heteropolymers). According to theinvention, “oligomers” refer to molecules containing from 2 to about 20monomers.

In the context of the invention, a “polyester containing material” or“polyester containing product” refers to a product, such as plasticproduct, comprising at least one polyester in crystalline,semi-crystalline or totally amorphous forms. In a particular embodiment,the polyester containing material refers to any item made from at leastone plastic material, such as plastic sheet, tube, rod, profile, shape,film, massive block etc., which contains at least one polyester, andpossibly other substances or additives, such as plasticizers, mineral ororganic fillers. In another particular embodiment, the polyestercontaining material refers to a plastic compound, or plasticformulation, in a molten or solid state, suitable for making a plasticproduct.

In the present description, “polyesters” encompass but is not limited topolyethylene terephthalate (PET), polytrimethylene terephthalate (PTT),polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate(PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylenesuccinate (PBS), polybutylene succinate adipate (PBSA), polybutyleneadipate terephthalate (PBAT), polyethylene furanoate (PEF),polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylenenaphthalate (PEN) and blends/mixtures of these polymers.

Novel Esterases with Improved Thermostability

The present invention provides novel esterases with improvedthermostability. More particularly, the inventors have developeddifferent ways to improve the stability of esterases at hightemperatures, and advantageously at temperature above 50° C., whichallow design of novel enzymes having superior properties for use inindustrial processes.

With the aim to improve the stability and/or activity of esterases inconditions where industrial degradation of plastic products can beperformed, the inventors have developed novel esterases derived from theesterase of SEQ ID N^(o) 1 that show high resistance to temperature. Theesterases of the invention are particularly suited to degrade plasticproduct containing PET.

The invention shows that, by creating new disulphide bridge(s) and/orsalt bridge(s) in the crystal structure of the protein; by reducingprotein mobility and/or solvent-excluded volume of intern cavities;and/or by reducing the N-terminal or C-terminal extremity, novelproteins are obtained which exhibit polyester degrading activity withimproved thermostability.

It is thus an object of the present invention to provide an esterasewhich (i) has at least 75%, 80%, 85%, 90%, 95% or 99% identity to thefull length amino acid sequence set forth in SEQ ID N^(o) 1, (ii)contains at least one amino acid modification as compared to SEQ IDN^(o) 1, (iii) has a polyester degrading activity and (iv) exhibitsincreased thermostability as compared to the esterase of SEQ ID N^(o) 1.

Within the context of the invention, the term “increasedthermostability” indicates an increased ability of the enzyme to resistto changes in its chemical and/or physical structure at hightemperatures, and more particularly at temperature between 50° C. and90° C., as compared to the esterase of SEQ ID N^(o) 1. Such an increaseis typically of about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more.Particularly, the esterases of the present invention may exhibit anincreased melting temperature (Tm) as compared to the esterase of SEQ IDN^(o) 1. In the context of the present invention, the meltingtemperature refers to the temperature at which half of theprotein/enzyme population considered is unfolded or misfolded.Typically, the esterase of the invention shows an increased Tm of about1° C., 2° C., 3° C., 4° C., 5° C., 10° C. or more, as compared to the Tmof the esterase of SEQ ID N^(o) 1.

In particular, the esterases of the present invention can have anincreased half-life at a temperature between 50° C. and 90° C., ascompared to the esterase of SEQ ID N^(o) 1. Furthermore, at suchtemperature, the esterases of the invention may exhibit greaterdegrading activity as compared to the esterase of SEQ ID N^(o) 1.

The thermostability of a protein may be evaluated by the one skilled inthe art, according to methods known per se in the art. For instance,thermostability can be assessed by analysis of the protein folding usingcircular dichroism. Alternatively or in addition, thermostability can beassessed by measuring the residual esterase activity and/or the residualpolyester depolymerization activity of the enzyme after incubation atdifferent temperatures. The ability to perform multiple rounds ofpolyester's depolymerization assays at different temperatures can alsobe evaluated. A rapid and valuable test may consist on the evaluation,by halo diameter measurement, of the enzyme ability to degrade a solidpolyester compound dispersed in an agar plate after incubation atdifferent temperatures. Preferably, a Differential Scanning Fluorimetry(DSF) is performed to assess the thermostability of a protein/enzyme.More particularly, the DSF may be used to quantify the change in thermaldenaturation temperature of a protein and thereby to determine itsmelting temperature (Tm). In the context of the invention, and unlessspecific indications, the Tm is measured using DSF as exposed in theexperimental part. In the context of the invention, comparisons of Tmare performed with Tm that are measured under same conditions (e.g. pH,nature and amount of polyesters, etc.).

In a particular embodiment, the variants of the invention have both animproved thermostability and an increased polyester degrading activityas compared to the esterase of SEQ ID N^(o) 1.

Within the context of the invention, the term “increased activity” or“increased degrading activity” indicates an increased ability of theenzyme to degrade a plastic product or material, and more particularly apolyester containing plastic product or material, as compared to theesterase of SEQ ID N^(o) 1. Such an increase is typically of about1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more. Particularly, theesterase variant has a polyester degrading activity at least 10% greaterthan the polyester degrading activity of the esterase of SEQ ID N^(o) 1,preferably at least 20%, 50%, 100%, 200%, 300%, or greater.

The activity of a protein may be evaluated by the one skilled in theart, according to methods known per se in the art. For instance, theactivity can be assessed by the measurement of the specific esteraseactivity rate, the measurement of the specific polyester'sdepolymerization activity rate, the measurement of the rate to degrade asolid polyester compound dispersed in an agar plate, or the measurementof the specific polyester's depolymerization activity rate in reactor.

Within the context of the invention, the terms “specific activity” or“specific degrading activity” designate the initial rate of oligomersand/or monomers released under suitable conditions of temperature, pHand buffer, when contacting the polyester containing plastic productwith a degrading enzyme, such as an esterase according to the invention.As an example, the specific activity of PET hydrolysis corresponds toμmol of PET hydrolysed/min or mg of equivalent TA produced/hour and permg of enzyme as determined in the linear part of the hydrolysis curve.

The ability of a protein to adsorb on a substrate may be evaluated bythe one skilled in the art, according to methods known per se in theart. For instance, the proteic content or the residual esteraseactivity, residual polyester's depolymerization activity, residualdegradation of a solid polyester compound dispersed in an agar plate, orresidual polyester's depolymerization activity in reactor can bemeasured from a solution containing the esterase of the invention andwherein the esterase has been previously incubated with a substrateunder suitable conditions where no enzymatic reaction can occur.

The esterases of the invention may comprise one or several modificationsas disclosed below.

In one embodiment, the esterase of the invention has at least 75%, 80%,85%, 90%, 95% or 99% identity to the full length amino acid sequence setforth in SEQ ID N^(o) 1 and comprises at least one additional disulphidebridge as compared to the esterase of SEQ ID N^(o) 1.

In a particular embodiment, the esterase variant comprises substitutionsat positions A172+A209, wherein the positions are numbered by referenceto the amino acid sequence set forth in SEQ ID N^(o) 1.

In another particular embodiment, the esterase variant comprises atleast one mutation at a position selected from V28 to G39, L82 and A103,wherein the positions are numbered by reference to the amino acidsequence set forth in SEQ ID N^(o) 1.

Particularly, the esterase variant exhibits a deletion of the amino acidresidues V28 to S34 of SEQ ID N^(o) 1, and substitutions as compared toSEQ ID N^(o) 1, at a position selected from G35, F36, G37, G38, G39, L82and/or A103, and more particularly substitutions consisting ofG35E/A+F36G+G37P+G38S+G39C, L82A and/or A103C.

Alternatively, the esterase variant exhibits a deletion of the aminoacids V33 to G39 of SEQ ID N^(o) 1, and substitutions as compared to SEQID N^(o) 1 consisting of V28E+S29G+R30P+L31S+S32C orV28A+S29G+R30P+L31S+S32C and substitution L82A and/or A103C.

Alternatively, the esterase variant comprises the replacement of theamino acids V28 to G39 of SEQ ID N^(o) 1 with the amino acid sequenceconsisting of E-G-P—S—C or A-G-P—S—C, and eventually substitution L82Aand/or A103C.

Alternatively, the esterase variant exhibits a deletion of the aminoacids V33 to G39 of SEQ ID N^(o) 1, and substitutions consisting ofV28E+S29G+R30P+L31S+S32C or V28A+S29G+R30P+L31S+S32C and substitutionsL82A, A103C, A172C and A209C.

In another particular embodiment, the esterase variant comprisessubstitutions at positions D203+S248, wherein the positions are numberedby reference to the amino acid sequence set forth in SEQ ID N^(o) 1.Preferably, the substitutions consist of D203C+S248C. In a particularembodiment, such esterase variant with substitutions at positionsD203+S248, further comprises at least one substitution at positionselected from E173, L202, N204 and F208. Preferably, the additionalsubstitutions are selected from E173R, E173A, F208W or F208I. Moreparticularly, the esterase variant comprises the substitutions selectedfrom D203C+S248C+E173R, D203C+S248C+E173A, D203C+S248C+F208W andD203C+S248C+F208I. In a particular embodiment, esterase variant withsubstitutions at positions D203+S248 further comprises at least twosubstitutions at positions selected from E173, L202, N204 and F208. Forinstance, the variant comprises at least the substitutionsD203C+S248C+E173R+N204D+L202R, F208W+D203C+S248C+E173A andF208I+D203C+S248C+E173A.

It is an object of the invention to provide an esterase having apolyester degrading activity which has at least 75%, 80%, 85%, 90%, 95%or 99% identity to the full length amino acid sequence set forth in SEQID N^(o) 1 and comprises, as compared to SEQ ID N^(o) 1, at least onemutation of an amino acid residue located in a void solvent-excludedcavity of the protein. Particularly, such mutation enables to reduce thesolvent-excluded volume of the cavity.

In a particular embodiment, the esterase variant comprises at least onesubstitution at a position selected from G53, L104, L107, L119, A121,L124, I54, M56, L70, L74, A127, V150, L152, L168, V170, P196, V198,V200, V219, Y220, T221, S223, W224, M225, L239, T252, N253, H256, byreference to SEQ ID N^(o) 1. Advantageously, the esterase variantcomprises at least two, three, four, five or more amino acidsubstitutions among said positions.

In an embodiment, at least one of said amino acids is replaced with abulkier (i.e., more voluminous) amino acid.

Advantageously, the esterase variant comprises at least one substitutionselected from G53A/I, I54L, M56I, L70M/I, L74M, L104M, L107M, L119M/A,A121V/I/M/Y, L124I/R/Q, A127V/I, V150I, L152I, L168I, V170I, V198I,V219I, Y220F/P/M, T221A/V/L/I/M, S223A, W2241/M, and T252S/D.

In a particular embodiment, the esterase variant comprises substitutionsat positions V170+F208. Advantageously, the esterase variant comprisessubstitutions V170I+F208W.

In a particular embodiment, the esterase variant comprises amino acidsubstitutions at positions belonging to at least two of the groups(i)-(v) below. Advantageously, the esterase variant comprises at leastsubstitution at a position in each group (i)-(v).

(i) G53, L104, L107, L119, A121, L124; (ii) I54, M56, L70, L74, A127,V150, L152, T221, M225;

(iii) V150, L152, L168, V170, V200, T221;

(iv) P196, V198, W224, T252, N253, H256; (v) V219, Y220, S223, L239.

It is an object of the invention to provide an esterase having apolyester degrading activity which has at least 75%, 80%, 85%, 90%, 95%or 99% identity to the full length amino acid sequence set forth in SEQID N^(o) 1, and comprises, as compared to SEQ ID N^(o) 1, at least oneadditional salt bridge. Preferably, the esterase comprises at least oneadditional surface salt bridge, located on the extern surface of theprotein structure.

To this aim, the esterase variant advantageously comprises at least oneamino acid substitution at a position selected from 51, Y4, Q5, R6, N9,S13, T16, S22, T25, Y26, S34, Y43, S48, T50, R72, S98, N105, R108, S113,N122, S145, K147, T160, N162, E173, S181, Q189, N190, S193, T194, D203,N204, S212, N213, N231, T233, R236, Q237, N241, N243, N254, R255, Q258.

Advantageously, the esterase variant has at least one salt bridgebetween two amino acids of said positions.

Most often, salt bridges are formed by interaction between an anioniccharge of either aspartic acid (D) or glutamic acid (E), and a cationiccharge of either lysine (K) or arginine (R). Accordingly, the saltbridge in the esterase of the invention is advantageously obtained bysubstituting at least one amino acid of at least one pair of targetamino acids as listed in Table 1, with D or E and/or K or R, dependingon the nature of the pair of target amino acid considered.

TABLE 1 Combination of pairs of amino acid positions (1^(st) and 2scd)to target to form salt bridge 1^(st) amino acid position 2scd amino acidposition S1 Q5 or N9 or S48 or N231or T233 or R236 or Q237 Y4 R6 or S48or T50 or N122 or N231 Q5 N9 or N231 or T233 or Q237 R6 S48 or T50 N9N231 or T233 or Q237 T16 N213 or S13 S22 Y43 or R72 T25 R72 Y26 Y43 orS113 S34 S98 or N105 Y43 S48 or S113 T50 S48 R108 N105 or S145 or N122S113 S48 N122 R6 or T50 or S145 K147 Y4 or N122 or S145 or N231 T160N190 N162 N190 or S193 or T194 E173 S181or D203 or N204 Q189 S181 orS193 S193 T160 or N190 or N254 or R255 T194 R255 S212 R72 N231 N122 R236T233 or N254 or Q258 Q237 N241 or N243 N243 R236 or N254 or Q258 Q258N241 or N254 or R255

Advantageously, when an amino acid residue of the targeted pair of aminoacids is R or K, solely the second amino acid of the targeted pair issubstituted with D or E. As an example, the esterase variant of theinvention may comprise the amino acid substitution Y4D and thereby mayexhibit a salt bridge between said mutated amino acid residue and R6. Inthe same way, when an amino acid residue of the targeted pair of aminoacids is D or E, solely the second amino acid of the targeted pair issubstituted with R or K. As an example, the esterase variant of theinvention may comprise the amino acid substitution N204R and thereby mayexhibit a salt bridge between said mutated amino acid residue and E173.

It is also an object of the invention to provide a esterase having apolyester degrading activity which has at least 75%, 80%, 85%, 90%, 95%or 99% identity to the full length amino acid sequence set forth in SEQID N^(o) 1, which comprises a suppression of at least one N- and/orC-terminal amino acid residue as compared to SEQ ID N^(o) 1, andpreferably, the suppression of at least one N-terminal amino acidresidue.

In a particular embodiment, the variant esterase of the inventioncomprises one or more amino acid substitution(s) as compared to SEQ IDN^(o) 1 at a position selected from T61, Y92 and V177, wherein thepositions are numbered by reference to the amino acid sequence set forthin SEQ ID N^(o) 1, and wherein the substitutions are different fromT61A/G, Y92A and V177A. Preferably, the variant esterase of theinvention comprises one or more amino acid substitution(s) as comparedto SEQ ID N^(o) 1, selected from V1771, Y92G/P, and T61M.

In another particular embodiment, the variant esterase of the inventioncomprises at least one substitution as compared to SEQ ID N^(o) 1 at theposition F208. According to the invention F208 may be substituted by anyone of the 19 other amino acids. Preferably, the substitution is F208W.

In another particular embodiment, the esterase variant comprises atleast two substitutions at positions selected from T61, Y92, V177 andF208 as compared to SEQ ID N^(o) 1, preferably at least twosubstitutions at positions F208 and Y92.

In a particular embodiment, the variant comprises at least thesubstitutions Y92P+F208W.

In another particular embodiment, the variant comprises at least thesubstitutions V170I+F208W.

According to the invention, the esterase variants may be furtherglycosylated to further increase the thermostability of the enzyme ascompared to the enzyme of SEQ ID N^(o) 1.

In a particular embodiment, the esterase variant comprises aglycosylated moiety on at least one asparagine residue of the enzyme,said asparagine residue being preferably at a position selected from N9,N143, N162, N204, N231, by reference to SEQ ID N^(o) 1, more preferablyselected from N9, N162 and N231. In an embodiment, esterase variantcomprises a glycosylated moiety on N9, N162 and N231.

For instance, a N-linked glycan moiety is attached to a nitrogen of atleast one of said asparagine residues.

Alternatively or in addition, the esterase variant of the invention mayfurther comprise one or more insertions of proline residue and/or one ormore deletions of glycine.

In a particular embodiment, the esterase variant of the inventioncomprises one or several modifications and/or mutations as listed above.

Novel Esterases with Both Improved Thermostability and Activity

It is a further object of the invention to provide novel esterases thatexhibit both increased thermostability and increased polyester degradingactivity as compared to the esterase of SEQ ID N^(o) 1.

It is thus another object of the invention to provide an esterase which(i) has at least 75%, 80%, 85%, 90%, 95% or 99% identity to the fulllength amino acid sequence set forth in SEQ ID N^(o) 1, (ii) contains atleast one amino acid modification as compared to SEQ ID N^(o) 1, and(iii) exhibits both an increased thermostability and an increasedactivity as compared to the esterase of SEQ ID N^(o) 1.

In a particular embodiment, the esterase variant comprises at least onemutation as disclosed above and at least one additional substitutionselected from F208I or F208W by reference to SEQ ID N^(o) 1.

In another particular embodiment, the variant comprises at least onesubstitution selected from T61M, Y92G/P, F208W, Y92P+F208W, andF208W+V170I and exhibits both an increased thermostability and anincreased activity as compared to the esterase of SEQ ID N^(o) 1.

In a particular embodiment, the variant comprises at least thesubstitution(s) selected from F208W+D203C+S248C and F208I+D203C+S248Cand exhibits both an increased thermostability and an increased activityas compared to the esterase of SEQ ID N^(o) 1.

Polyester Degrading Activity

It is an object of the invention to provide new enzymes having anesterase activity. In a particular embodiment, the enzyme of theinvention further exhibits a cutinase activity.

In a particular embodiment, the esterase of the invention has apolyester degrading activity, preferably a polyethylene terephthalate(PET) degrading activity.

In another particular embodiment, the esterase of the invention also hasa PBAT degrading activity.

Advantageously, the esterase variant of the invention exhibits apolyester degrading activity at least in a range of temperatures from20° C. to 90° C., preferably from 40° C. to 80° C., more preferably from50° C. to 70° C., even more preferably from 60° C. to 70° C., even morepreferably at 65° C. In a particular embodiment, the esterase variant ofthe invention exhibits a polyester degrading activity at 70° C. Inanother particular embodiment, the polyester degrading activity is stillmeasurable at a temperature between 60° C. and 90° C.

In a particular embodiment, the esterase variant of the invention has anincreased half-life at a given temperature, compared to the esterase ofSEQ ID N^(o) 1, and more particularly at a temperature between 40° C.and 80° C., more preferably between 50° C. and 70° C., even morepreferably between 60° C. and 70° C., even more preferably at 65° C. Ina particular embodiment, the esterase variant has a half-life at 65° C.at least 5% greater than the half-life of the esterase of SEQ ID N^(o)1, preferably at least 10%, 20%, 50%, 100%, 200%, 300%, or greater.

In another particular embodiment, the esterase variant of the inventionhas an increased melting temperature (Tm), as compared to the esteraseof SEQ ID N^(o) 1. Advantageously, the esterase variant of the inventionhas an melting temperature (Tm) increased of about 1° C., 2° C., 3° C.,4° C., 5° C., 10° C. or more as compared to the esterase of SEQ ID N^(o)1.

In a particular embodiment, the esterase variant of the inventionexhibits a measurable esterase activity at least in a range of pH from 5to 11, preferably in a range of pH from 6 to 9, more preferably in arange of pH from 6.5 to 9, even more preferably in a range of pH from6.5 to 8.

Nucleic Acids, Expression Cassette, Vector

It is a further object of the invention to provide a nucleic acidencoding an esterase as defined above.

As used herein, the term “nucleic acid”, “nucleic sequence,”“polynucleotide”, “oligonucleotide” and “nucleotide sequence” are usedinterchangeably and refer to a sequence of deoxyribonucleotides and/orribonucleotides. The nucleic acids can be DNA (cDNA or gDNA), RNA, or amixture of the two. It can be in single stranded form or in duplex formor a mixture of the two. It can be of recombinant, artificial and/orsynthetic origin and it can comprise modified nucleotides, comprisingfor example a modified bond, a modified purine or pyrimidine base, or amodified sugar. The nucleic acids of the invention can be in isolated orpurified form, and made, isolated and/or manipulated by techniques knownper se in the art, e.g., cloning and expression of cDNA libraries,amplification, enzymatic synthesis or recombinant technology. Thenucleic acids can also be synthesized in vitro by well-known chemicalsynthesis techniques, as described in, e.g., Belousov (1997) NucleicAcids Res. 25:3440-3444.

The invention also encompasses nucleic acids which hybridize, understringent conditions, to a nucleic acid encoding an esterase as definedabove. Preferably, such stringent conditions include incubations ofhybridization filters at about 42° C. for about 2.5 hours in 2×SSC/0.1%SDS, followed by washing of the filters four times of 15 minutes in1×SSC/0.1% SDS at 65° C. Protocols used are described in such referenceas Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel (CurrentProtocols in Molecular Biology (1989)).

The invention also encompasses nucleic acids encoding an esterase of theinvention, wherein the sequence of said nucleic acids, or a portion ofsaid sequence at least, has been engineered using optimized codon usage.

Alternatively, the nucleic acids according to the invention may bededuced from the sequence of the esterase according to the invention andcodon usage may be adapted according to the host cell in which thenucleic acids shall be transcribed. These steps may be carried outaccording to methods well known to one skilled in the art and some ofwhich are described in the reference manual Sambrook et al. (Sambrook etal., 2001).

Nucleic acids of the invention may further comprise additionalnucleotide sequences, such as regulatory regions, i.e., promoters,enhancers, silencers, terminators, signal peptides and the like that canbe used to cause or regulate expression of the polypeptide in a selectedhost cell or system.

The present invention further relates to an expression cassettecomprising a nucleic acid according to the invention operably linked toone or more control sequences that direct the expression of said nucleicacid in a suitable host cell. Typically, the expression cassettecomprises, or consists of, a nucleic acid according to the inventionoperably linked to a control sequence such as transcriptional promoterand/or transcription terminator. The control sequence may include apromoter that is recognized by a host cell or an in vitro expressionsystem for expression of a nucleic acid encoding an esterase of thepresent invention. The promoter contains transcriptional controlsequences that mediate the expression of the enzyme. The promoter may beany polynucleotide that shows transcriptional activity in the host cellincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell. The control sequence mayalso be a transcription terminator, which is recognized by a host cellto terminate transcription. The terminator is operably linked to the3′-terminus of the nucleic acid encoding the esterase. Any terminatorthat is functional in the host cell may be used in the presentinvention. Typically, the expression cassette comprises, or consists of,a nucleic acid according to the invention operably linked to atranscriptional promoter and a transcription terminator.

The invention also relates to a vector comprising a nucleic acid or anexpression cassette as defined above.

The term “vector” refers to DNA molecule used as a vehicle to transferrecombinant genetic material into a host cell. The major types ofvectors are plasmids, bacteriophages, viruses, cosmids, and artificialchromosomes. The vector itself is generally a DNA sequence that consistsof an insert (a heterologous nucleic acid sequence, transgene) and alarger sequence that serves as the “backbone” of the vector. The purposeof a vector which transfers genetic information to the host is typicallyto isolate, multiply, or express the insert in the target cell. Vectorscalled expression vectors (expression constructs) are specificallyadapted for the expression of the heterologous sequences in the targetcell, and generally have a promoter sequence that drives expression ofthe heterologous sequences encoding a polypeptide. Generally, theregulatory elements that are present in an expression vector include atranscriptional promoter, a ribosome binding site, a terminator, andoptionally present operator. Preferably, an expression vector alsocontains an origin of replication for autonomous replication in a hostcell, a selectable marker, a limited number of useful restriction enzymesites, and a potential for high copy number. Examples of expressionvectors are cloning vectors, modified cloning vectors, specificallydesigned plasmids and viruses. Expression vectors providing suitablelevels of polypeptide expression in different hosts are well known inthe art. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced.

It is another object of the invention to provide a host cell comprisinga nucleic acid, an expression cassette or a vector as described above.The present invention thus relates to the use of a nucleic acid,expression cassette or vector according to the invention to transform,transfect or transduce a host cell. The choice of the vector willtypically depend on the compatibility of the vector with the host cellinto which it must be introduced.

According to the invention, the host cell may be transformed,transfected or transduced in a transient or stable manner. Theexpression cassette or vector of the invention is introduced into a hostcell so that the cassette or vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector. The term“host cell” also encompasses any progeny of a parent host cell that isnot identical to the parent host cell due to mutations that occur duringreplication. The host cell may be any cell useful in the production of avariant of the present invention, e.g., a prokaryote or a eukaryote. Theprokaryotic host cell may be any Gram-positive or Gram-negativebacterium. The host cell may also be an eukaryotic cell, such as ayeast, fungal, mammalian, insect or plant cell. In a particularembodiment, the host cell is selected from the group of Escherichiacoli, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces,Pichia or Yarrowia.

The nucleic acid, expression cassette or expression vector according tothe invention may be introduced into the host cell by any method knownby the skilled person, such as electroporation, conjugation,transduction, competent cell transformation, protoplast transformation,protoplast fusion, biolistic “gene gun” transformation, PEG-mediatedtransformation, lipid-assisted transformation or transfection,chemically mediated transfection, lithium acetate-mediatedtransformation, liposome-mediated transformation.

Optionally, more than one copy of a nucleic acid, cassette or vector ofthe present invention may be inserted into a host cell to increaseproduction of the variant.

In a particular embodiment, the host cell is a recombinantmicroorganism. The invention indeed allows the engineering ofmicroorganisms with improved capacity to degrade polyester containingmaterial. For instance, the sequence of the invention may be used tocomplement a wild type strain of a fungus or bacterium already known asable to degrade polyester, in order to improve and/or increase thestrain capacity.

Production of Esterase Variant

It is another object of the invention to provide a method of producingthe esterase variant of the invention, comprising expressing a nucleicacid encoding the esterase and optionally recovering the esterase.

In particular, the present invention relates to in vitro methods ofproducing an esterase of the present invention comprising (a) contactinga nucleic acid, cassette or vector of the invention with an in vitroexpression system; and (b) recovering the esterase produced. In vitroexpression systems are well-known by the person skilled in the art andare commercially available.

Preferably, the method of production comprises

(a) culturing a host cell that comprises a nucleic acid encoding anesterase of the invention under conditions suitable to express thenucleic acid; and optionally(b) recovering said esterase from the cell culture.

Advantageously, the host cell is a recombinant Bacillus, recombinant E.coli, recombinant Aspergillus, recombinant Trichoderma, recombinantStreptomyces, recombinant Saccharomyces, recombinant Pichia orrecombinant Yarrowia lipolytica.

The host cells are cultivated in a nutrient medium suitable forproduction of polypeptides, using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymeto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium, from commercial suppliers or preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection).

If the esterase is excreted into the nutrient medium, the esterase canbe recovered directly from the culture supernatant. Conversely, theesterase can be recovered from cell lysates or after permeabilisation.The esterase may be recovered using any method known in the art. Forexample, the esterase may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. Optionally, the esterase may be partially or totallypurified by a variety of procedures known in the art including, but notlimited to, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction to obtainsubstantially pure polypeptides.

The esterase may be used as such, in purified form, either alone or incombinations with additional enzymes, to catalyze enzymatic reactionsinvolved in the degradation and/or recycling of a polyester containingmaterial, such as plastic products containing polyester. The esterasemay be in soluble form, or on solid phase. In particular, it may bebound to cell membranes or lipid vesicles, or to synthetic supports suchas glass, plastic, polymers, filter, membranes, e.g., in the form ofbeads, columns, plates and the like.

Composition

It is a further object of the invention to provide a compositioncomprising an esterase or a host cell of the invention. In the contextof the invention, the term “composition” encompasses any kind ofcompositions comprising an esterase of the invention. In a particularembodiment, the esterase is in isolated or at least partially purifiedform.

The composition may be liquid or dry, for instance in the form of apowder. In some embodiments, the composition is a lyophilisate. Forinstance, the composition may comprise the esterase and/or recombinantcells encoding the esterase of the invention or extract thereof, andoptionally excipients and/or reagents etc. Appropriate excipientsencompass buffers commonly used in biochemistry, agents for adjustingpH, preservatives such as sodium benzoate, sodium sorbate or sodiumascorbate, conservatives, protective or stabilizing agents such asstarch, dextrin, arabic gum, salts, sugars e.g. sorbitol, trehalose orlactose, glycerol, polyethyleneglycol, polyethene glycol, polypropyleneglycol, propylene glycol, sequestering agent such as EDTA, reducingagents, amino acids, a carrier such as a solvent or an aqueous solution,and the like. The composition of the invention may be obtained by mixingthe esterase with one or several excipients.

The composition of the invention may comprise from 0.1% to 99.9%,preferably from 0.1% to 50%, more preferably from 0.1% to 30%, even morepreferably from 0.1% to 5% by weight of the esterase of the inventionand from 0.1% to 99.9%, preferably from 50% to 99.9%, more preferablyfrom 70% to 99.9%, even more preferably from 95% to 99.9% by weight ofexcipient(s). A preferred composition comprises between 0.1 and 5% byweight of the esterase of the invention.

In a particular embodiment, the composition may further compriseadditional polypeptide(s) exhibiting an enzymatic activity. The amountsof esterase of the invention will be easily adapted by those skilled inthe art depending e.g., on the nature of the polyester containingmaterial to degrade and/or the additional enzymes/polypeptides containedin the composition.

In a particular embodiment, the esterase of the invention is solubilizedin an aqueous medium together with one or several excipients, especiallyexcipients which are able to stabilize or protect the polypeptide fromdegradation. For instance, the esterase of the invention may besolubilized in water, eventually with additional components, such asglycerol, sorbitol, dextrin, starch, glycol such as propanediol, salt,etc. The resulting mixture may then be dried so as to obtain a powder.Methods for drying such mixture are well known to the one skilled in theart and include, without limitation, lyophilisation, freeze-drying,spray-drying, supercritical drying, down-draught evaporation, thin-layerevaporation, centrifugal evaporation, conveyer drying, fluidized beddrying, drum drying or any combination thereof.

In a further particular embodiment, the composition of the inventioncomprises at least one recombinant cell expressing an esterase of theinvention, or an extract thereof. An “extract of a cell” designates anyfraction obtained from a cell, such as cell supernatant, cell debris,cell walls, DNA extract, enzymes or enzyme preparation or anypreparation derived from cells by chemical, physical and/or enzymatictreatment, which is essentially free of living cells. Preferred extractsare enzymatically-active extracts. The composition of the invention maycomprise one or several recombinant cells of the invention or extractthereof, and optionally one or several additional cells.

In a particular embodiment, the composition consists or comprises alyophilized culture medium of a recombinant microorganism expressing andexcreting an esterase of the invention. In a particular embodiment, thepowder comprises the esterase of the invention and astabilizing/solubilizing amount of glycerol, sorbitol or dextrin, suchas maltodextrine and/or cyclodextrine, starch, glycol such aspropanediol, and/or salt.

Use of the Esterase of the Invention

It is a further object of the invention to provide methods using anesterase of the invention for degrading in aerobic or anaerobicconditions and/or recycling polyester containing material, as plasticproducts made of or containing polyesters. The variant esterases of theinvention are particularly useful for degrading a plastic productcomprising PET.

It is therefore an object of the invention to use an esterase of theinvention, or corresponding recombinant cell or extract thereof, orcomposition for the enzymatic degradation of a polyester containingmaterial, such as a PET containing material.

It is another object of the invention to provide a method for degradinga plastic product containing at least one polyester, wherein the plasticproduct is contacted with an esterase or host cell or composition of theinvention, thereby degrading the plastic product. Advantageously,polyester(s) of the polyester containing material is (are) depolymerizedup to monomers and/or oligomers.

In an embodiment of the method of degradation, at least one polyester isdegraded to yield repolymerizable monomers and/or oligomers, which areadvantageously retrieved in order to be reused.

In an embodiment, polyester(s) of the polyester containing material is(are) fully degraded.

In a particular embodiment, the plastic product comprises at least onepolyester selected from polyethylene terephthalate (PET),polytrimethylene terephthalate (PTT), polybutylen terephthalate (PBT),polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA),polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylenesuccinate adipate (PBSA), polybutylene adipate terephthalate (PBAT),polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethyleneadipate) (PEA), polyethylene naphthalate (PEN) and blends/mixtures ofthese materials, preferably polyethylene terephthalate. In a preferredembodiment, the polyester containing material comprises PET, and atleast monomers such as monoethylene glycol or terephthalic acid, and/oroligomers such as methyl-2-hydroxyethyl terephthalate (MHET),bis(2-hydroxyethyl) terephthalate (BHET), 2-hydroxyethyl benzoate (HEB)and dimethyl terephthalate (DMT) are recovered for recycling ormethanisation for instance.

The invention also relates to a method of producing monomers and/oroligomers from a polyester containing material, comprising exposing apolyester containing material to an esterase of the invention, orcorresponding recombinant cell or extract thereof, or composition, andoptionally recovering monomers and/or oligomers. The method of theinvention is particularly useful for producing monomers selected frommonoethylene glycol and terephthalic acid, and/or oligomers selectedfrom methyl-2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl)terephthalate (BHET), 2-hydroxyethyl benzoate (HEB) and dimethylterephthalate (DMT).

The time required for degrading a polyester containing material may varydepending on the polyester containing material itself (i.e., nature andorigin of the plastic product, its composition, shape etc.), the typeand amount of esterase used, as well as various process parameters(i.e., temperature, pH, additional agents, etc.). One skilled in the artmay easily adapt the process parameters to the polyester containingmaterial.

Advantageously, the degrading process is implemented at a temperaturecomprised between 20° C. and 90° C., preferably between 40° C. and 80°C., more preferably between 50° C. and 70° C., more preferably between60° C. and 70° C., even more preferably at 65° C. In another particularembodiment, the degrading process is implemented at 70° C. Moregenerally, the temperature is maintained below an inactivatingtemperature, which corresponds to the temperature at which the esteraseis inactivated and/or the recombinant microorganism does no moresynthesize the esterase. Particularly, the temperature is maintainedbelow the glass transition temperature (Tg) of the polyester in thepolyester containing material. More particularly, the process isimplemented in a continuous way, at a temperature at which the esterasecan be used several times and/or recycled.

Advantageously, the degrading process is implemented at a pH comprisedbetween 5 and 11, preferably at a pH between 6 and 9, more preferably ata pH between 6.5 and 9, even more preferably at a pH between 6.5 to 8.

In a particular embodiment, the polyester containing material may bepretreated prior to be contacted with the esterase, in order tophysically change its structure, so as to increase the surface ofcontact between the polyester and the variant of the invention.

Optionally, monomers and/or oligomers resulting from thedepolymerization may be recovered, sequentially or continuously. Asingle type of monomers and/or oligomers or several different types ofmonomers and/or oligomers may be recovered, depending on the startingpolyester containing material.

The recovered monomers and/or oligomers may be further purified, usingall suitable purifying methods and conditioned in a re-polymerizableform. Examples of purifying methods include stripping process,separation by aqueous solution, steam selective condensation, filtrationand concentration of the medium after the bioprocess, separation,distillation, vacuum evaporation, extraction, electrodialysis,adsorption, ion exchange, precipitation, crystallization, concentrationand acid addition dehydration and precipitation, nanofiltration, acidcatalyst treatment, semi continuous mode distillation or continuous modedistillation, solvent extraction, evaporative concentration, evaporativecrystallization, liquid/liquid extraction, hydrogenation, azeotropicdistillation process, adsorption, column chromatography, simple vacuumdistillation and microfiltration, combined or not.

The repolymerizable monomers and/or oligomers may then be reused forinstance to synthesize polyesters. Advantageously, polyesters of samenature are repolymerized. However, it is possible to mix the recoveredmonomers and/or oligomers with other monomers and/or oligomers, in orderfor instance to synthesize new copolymers. Alternatively, the recoveredmonomers may be used as chemical intermediates in order to produce newchemical compounds of interest.

The invention also relates to a method of surface hydrolysis or surfacefunctionalization of a polyester containing material, comprisingexposing a polyester containing material to an esterase of theinvention, or corresponding recombinant cell or extract thereof, orcomposition. The method of the invention is particularly useful forincreasing hydrophilicity, or water absorbency, of a polyester material.Such increased hydrophilicity may have particular interest in textilesproduction, electronics and biomedical applications.

It is a further object of the invention to provide a polyestercontaining material in which an esterase of the invention and/or arecombinant microorganism expressing and excreting said esterase is/areincluded. In a particular embodiment, such polyester containing materialmay be a plastic compound. It is thus an object of the invention toprovide a plastic compound containing an esterase of the inventionand/or a recombinant cell and/or a composition or extract thereof; andat least one polyester. In a preferred embodiment, the polyester is PET.

EXAMPLES Example 1—Construction, Expression and Purification ofEsterases

Construction

The esterase variants have been generated using the plasmidicconstruction pET26b-LCC-His. This plasmid consists in cloning a geneencoding the esterase of SEQ ID N^(o) 1, optimized for Escherichia coliexpression between NdeI and XhoI restriction sites. Two site directedmutagenesis kits have been used according to the recommendations of thesupplier, in order to generate the esterase variants: QuikChange IISite-Directed Mutagenesis kit and QuikChange Lightning MultiSite-Directed from Agilent (Santa Clara, Calif., USA).

Expression and Purification of the Esterases

The strains Stellar™ (Clontech, Calif., USA) and E. coli One Shot® BL21DE3 (Life technologies, Carlsbad, Calif., USA) have been successivelyemployed to perform the cloning and recombinant expression in 50 mLLB-Miller medium or ZYM auto inducible medium (Studier et al.,2005—Prot. Exp. Pur. 41, 207-234). The induction in LB-Miller medium hasbeen performed at 16° C., with 0.5 mM of isopropylβ-D-1-thiogalactopyranoside (IPTG, Euromedex, Souffelweyersheim,France). The cultures have been stopped by centrifugation (8000 rpm, 20minutes at 10° C.) in an Avanti J-26 XP centrifuge (Beckman Coulter,Brea, USA). The cells have been suspended in 20 mL of Talon buffer(Tris-HCl 20 mM, NaCl 300 mM, pH 8). Cell suspension was then sonicatedduring 2 minutes with 30% of amplitude (2 sec ON and 1 sec OFF cycles)by FB 705 sonicator (Fisherbrand, Illkirch, France). Then, a step ofcentrifugation has been realized: 30 minutes at 11000 rpm, 10° C. in anEppendorf centrifuge. The soluble fraction has been collected andsubmitted to affinity chromatography. This purification step has beencompleted with Talon® Metal Affinity Resin (Clontech, Calif., USA).Protein elution has been carried out with gradient of Talon buffersupplemented with imidazole. Purified protein has been dialyzed againstTalon buffer then quantified using Bio-Rad protein assay according tomanufacturer instructions (Lifescience Bio-Rad, France) and stored at+4° C.

Example 2—Evaluation of the Thermostability of the Esterases of theInvention

The thermostability of the esterase variants has been determined andcompared to the thermostability of the esterase of SEQ ID N^(o) 1.

Different methodologies have been used to estimate thermostability:

(1) Circular dichroism of proteins in solution;(2) Residual esterase activity after protein incubation in givenconditions of temperatures, times and buffers;(3) Residual polyester's depolymerization activity after proteinincubation in given conditions of temperatures, times and buffers;(4) Ability to degrade a solid polyester compound (such as PET or PBATor analogues) dispersed in an agar plate, after protein incubation ingiven conditions of temperatures, times and buffers;(5) Ability to perform multiple rounds of polyester's depolymerizationassays in given conditions of temperatures, buffers, proteinconcentrations and polyester concentrations;

(6) Differential Scanning Fluorimetry (DSF).

Details on the protocol of such methods are given below.

2.1 Circular Dichroism

Circular dichroism (CD) has been performed with a Jasco 815 device(Easton, USA) to compare the fusion temperature (T_(m)) of the esteraseof SEQ ID N^(o) 1 and the esterase variants of the invention. The T_(m)corresponds to the temperature at which 50% of the protein isdenaturated.

Technically 4004, protein sample was prepared at 0.5 mg/mL in Talonbuffer and used for CD. A first scan from 280 to 190 nm was realized todetermine two maxima intensities of CD corresponding to the correctfolding of the protein. A second scan was then performed from 25° C. to110° C., at length waves corresponding to such maximal intensities andproviding specific curves (sigmoid 3 parameters y=a/(1+e{circumflex over( )}((x−x0)/b))) that were analyzed by Sigmaplot version 11.0 software,the Tm is determined when x=x0. The T_(m) obtained reflects thethermostability of the given protein. The higher the T_(m) is, the morestable the variant is at high temperature.

2.2 Residual Esterase Activity

1 mL of a solution of 40 mg/L (in Talon buffer) of the esterase of SEQID N^(o) 1 or of an esterase variant was incubated at differenttemperatures (65, 70, 75, 80 and 90° C.) during 10 days. Regularly, asample, was taken, diluted 1 to 500 times in a 0.1M potassium phosphatebuffer pH 8.0 and para nitro phenol-butyrate (pNP-B) assay was realized.20 μL of sample are mixed with 175 μL of 0.1M potassium phosphate bufferpH 8.0 and 5 μL of pNP-B solution in 2-methyl-2 butanol (40 mM).Enzymatic reaction was performed at 30° C. under agitation, during 15minutes and absorbance at 405 nm was acquired by microplatespectrophotometer (Versamax, Molecular Devices, Sunnyvale, Calif., USA).Activity of pNP-B hydrolysis (initial velocity expressed in μmol ofpNPB/min) was determined using a standard curve for the liberated paranitro phenol in the linear part of the hydrolysis curve. The half-lifeof the enzyme at a given temperature corresponds to the time necessaryto lose 50% of the initial activity.

2.3 Residual Polyester Depolymerizing Activity

Crushed Cristal preform were immersed in liquid nitrogen and weremicronized using an Ultra Centrifugal Mill ZM 200 system to a finepowder<500 μm size. Then, the obtained powder was sieved. The fractionwith a size between 250 μm and 500 μm only has been used for thedepolymerization test. The crystallinity of this fraction was measuredat 11.5% using a Mettler Toledo DSC 3 with heating rate of 10° C./min.

10 mL of a solution of 40 mg/L (in Talon buffer) of the esterase of SEQID N^(o) 1 and of an esterase variant respectively were incubated atdifferent temperatures (65, 70, 75, 80 and 90° C.) during 10 to 30 days.Regularly, a 1 mL sample was taken, and transferred into a bottlecontaining 100 mg of amorphous PET micronized at 250-500 μm and 49 mL of0.1M potassium phosphate buffer pH 8.0 and incubated at 65° C. 150 μL ofbuffer were sampled regularly. When required, samples were diluted in0.1 M potassium phosphate buffer pH 8. Then, 150 μL of methanol and 6.5μL of HCl 6 N were added to 150 μL of sample or dilution. After mixingand filtering on 0.45 μm syringe filter, samples were loaded on UHPLC tomonitor the liberation of terephthalic acid (TA), MHET and BHET.Chromatography system used was an Ultimate 3000 UHPLC system (ThermoFisher Scientific, Inc. Waltham, Mass., USA) including a pump module, anautosampler, a column oven thermostated at 25° C., and an UV detector at240 nm. The column used was a Discovery® HS C18 HPLC Column (150×4.6 mm,5 μm, equipped with precolumn, Supelco, Bellefonte, USA). TA, MHET andBHET were separated using a gradient of MeOH (30% to 90%) in 1 mM ofH2504 at 1 mL/min. Injection was 20 μL of sample. TA, MHET and BHET weremeasured according to standard curves prepared from commercial TA andBHET and in house synthetized MHET in the same conditions than samples.Activity of PET hydrolysis (μmol of PET hydrolysed/min or mg ofequivalent TA produced/hour) was determined in the linear part of thehydrolysis curve. Equivalent TA corresponds to the sum of TA measuredand of TA contained in measured MHET and BHET. The half-life of theenzyme at a given temperature corresponds to the time required to lose50% of the initial activity.

2.4 Degradation of a Polyester Under Solid Form

20 μL of enzyme preparation was deposited in a well created in an agarplate containing PET. Preparation of agar plates was realized bysolubilizing 500 mg of PET solubilized in HFIP, and this medium ispoured in a 250 mL aqueous solution. After HFIP evaporation at 52° C.,the solution was mixed v/v with 0.2 M potassium phosphate buffer pH 8containing 3% agar. Around 30 mL of the mixture is used to prepare eachomnitray and stored at 4° C.

The diameter of the halo formed due to the polyester degradation wasmeasured after 24 hours at 60 or 65° C. The half-life of the enzyme at agiven temperature corresponds to the time required to decrease by a2-fold factor the diameter of the halo.

2.5 Multiple Rounds of Polyester's Depolymerization

The ability of the esterase to perform successive rounds of polyester'sdepolymerization assays was evaluated in an enzymatic reactor. A Minibio500 bioreactor (Applikon Biotechnology B.V., Delft, The Netherlands) wasstarted with 3 g of amorphous PET and 100 mL of 10 mM potassiumphosphate buffer pH 8 containing 3 mg of LC-esterase. Agitation was setat 250 rpm using a marine impeller. Bioreactor was thermostated at 65°C. by immersion in an external water bath. pH was regulated at 8 byaddition of KOH at 3 M. The different parameters (pH, temperature,agitation, addition of base) were monitored thanks to BioXpert softwareV2.95. 1.8 g of amorphous PET were added every 20 h. 500 μL of reactionmedium was sampled regularly.

Amount of TA, MHET and BHET was determined by HPLC, as described inexample 2.3. Amount of EG was determined using an Aminex HPX-87K column(Bio-Rad Laboratories, Inc, Hercules, Calif., United States)thermostated at 65° C. Eluent was K₂HPO₄ 5 mM at 0.6 mL.min⁻¹. Injectionwas 20 μL. Ethylene glycol was monitored using refractometer.

The percentages of hydrolysis were calculated based on the ratio ofmolar concentration at a given time (TA+MHET+BHET) versus the totalamount of TA contained in the initial sample, or based on the ratio ofmolar concentration at a given time (EG+MHET+2×BHET) versus the totalamount of EG contained in the initial sample. Rate of degradation iscalculated in mg of total liberated TA per hour or in mg of total EG perhour.

Half-life of enzyme was evaluated as the incubation time required toobtain a loss of 50% of the degradation rate.

2.6 Differential Scanning Fluorimetry (DSF)

DSF was used to evaluate the thermostability of the wild-type protein(SEQ ID N^(o) 1) and variants thereof by determining their meltingtemperature (Tm), temperature at which half of the protein population isunfolded. Protein samples were prepared at a concentration of 14 μM (0.4mg/mL) and stored in buffer A consisting of 20 mM Tris HCl pH 8.0, 300mM NaCl. The SYPRO orange dye 5000× stock solution in DMSO was firstdiluted to 250× in water. Protein samples were loaded onto a white clear96-well PCR plate (Bio-Rad cat # HSP9601) with each well containing afinal volume of 25 μl. The final concentration of protein and SYPROOrange dye in each well were 5 μM (0.14 mg/ml) and 10× respectively.Loaded volumes per well were as follow: 15 μL of buffer A, 9 μL of the0.4 mg/mL protein solution and 1 μL of the 250× Sypro Orange dilutedsolution. The PCR plates were then sealed with optical quality sealingtape and spun at 2000 rpm for 1 min at room temperature. DSF experimentswere then carried out using a CFX96 real-time PCR system set to use the450/490 excitation and 560/580 emission filters. The samples were heatedfrom 25 to 100° C. at the rate of 1.1° C./min. A single fluorescencemeasurement was taken every 0.3° C. Melting temperatures were determinedby performing a curve fit to the Boltzmann equation.

Wild-type protein and variants were then compared based on their Tmvalues. Due to high reproducibility between experiments on the sameprotein from different productions, a ΔTm of 0.8° C. was considered assignificant to compare variants. Tm values correspond to the average ofat least 2 measurements.

Compared thermostabilities of esterase variants of the invention areshown in Table 2 below, expressed in Tm values and evaluated accordingto Example 2.6. The gain of Tm as compared to the esterase of SEQ IDN^(o) 1 is indicated in brackets.

TABLE 2 Tm of the esterases of the invention Variant of the invention Tmof variant of the invention D203C + S248C 94.20° C. +/− 0.00° C. (+9.50°C.) D203C + S248C + E173A 93.45° C. +/− 0.21° C. (+8.75° C.) D203C +S248C + E173R 92.25° C. +/− 0.21° C. (+7.55° C.) D203C + S248C + E173R +92.80° C. +/− 0.17° C. (+8.10° C.) N204D + L202R F208W 85.90° C. +/−0.17° C. (+1.20° C.) Y92P 85.80° C. +/− 0.00° C. (+1.10° C.) V177I86.10° C. +/− 0.30° C. (+1.40° C.) T61M 87.40° C. +/− 0.17° C. (+2.70°C.) Y92G 87.00° C. +/− 0.00° C. (+2.30° C.) Y92P + F208W 86.60° C. +/−0.17° C. (+1.90° C.) F208W + V170I 85.80° C. +/− 0.00° C. (+1.10° C.)F208W + D203C + S248C 94.80° C. +/− 0.00° C. (+10.10° C.) F208I +D203C + S248C 90.90° C. +/− 0.00° C. (+6.20° C.) F208W + D203C + S248C +94.20° C. +/− 0.00° C. (+9.50° C.) E173A F208I + D203C + S248C + 89.40°C. +/− 0.00° C. (+4.70° C.) E173A

Example 3—Evaluation of the Thermostability and Activity of EsteraseVariants of the Invention

The specific degrading activity of esterase variants of the inventionhas been evaluated on PET and compared with the specific degradingactivity of the esterase of SEQ ID N^(o) 1.

100 mg of amorphous PET were weighted and introduced in a 100 mL glassbottle. 1 mL of esterase preparation (as reference control) or ofvariant preparation, respectively, prepared at 0.02 or 0.03 mg/mL inTalon buffer (Tris-HCl 20 mM, NaCl 0.3M, pH 8) and introduced in theglass bottle. Finally, 49 mL of 0.1 M potassium phosphate buffer pH 8was added.

The depolymerization started by incubating each glass bottle at 65° C.and 150 rpm in a Max Q 4450 incubator (Thermo Fisher Scientific, Inc.Waltham, Mass., USA).

The initial rate of depolymerization reaction, in mg of equivalent TAgenerated/hour, was determined by samplings performed at different timeduring the first 24 hours and analyzed by Ultra High Performance LiquidChromatography (UHPLC). If necessary, samples were diluted in 0.1 Mpotassium phosphate buffer pH 8. Then, 150 μL of methanol and 6.5 μL ofHCl 6 N were added to 150 μL of sample or dilution. After mixing andfiltering on 0.45 μm syringe filter, samples were loaded on UHPLC tomonitor the liberation of terephthalic acid (TA), MHET and BHET.Chromatography system used was an Ultimate 3000 UHPLC system (ThermoFisher Scientific, Inc. Waltham, Mass., USA) including a pump module, anautosampler, a column oven thermostated at 25° C., and an UV detector at240 nm. The column used was a Discovery® HS C18 HPLC Column (150×4.6 mm,5 μm, equipped with precolumn, Supelco, Bellefonte, USA). TA, MHET andBHET were separated using a gradient of MeOH (30% to 90%) in 1 mM ofH2504 at 1 mL/min. Injection was 20 μL of sample. TA, MHET and BHET weremeasured according to standard curves prepared from commercial TA andBHET and in house synthetized MHET in the same conditions than samples.The specific degrading activity of PET (mg of equivalent TA/hour/mg ofenzyme) was determined in the linear part of the hydrolysis curve.

The results for both specific degrading activity and thermostability ofthe esterase variants of the invention are shown in Table 3.

The specific degrading activity of the esterase of SEQ ID N^(o) 1 isused as a reference and considered as 100% degrading activity. Thedegrading activity is measured as exposed in example 3 (mg of equivalentTA/hour/mg of enzyme). Equivalent TA corresponds to the sum of TAmeasured and of TA contained in measured MHET and BHET. Thethermostability is expressed in Tm values (measured according to example2.6) and the gain of Tm as compared to the Tm of the esterase of SEQ IDN^(o) 1 is noted in brackets.

TABLE 3 Specific activity and Tm of the esterases of the inventionSpecific Variants of degrading the invention activity Tm of the variantof the invention F208W 143% 85.90° C. +/− 0.17° C. (+1.20° C.) Y92P 177%85.80° C. +/− 0.00° C. (+1.10° C.) T61M 121% 87.40° C. +/− 0.17° C.(+2.70° C.) Y92G 120% 87.00° C. +/− 0.00° C. (+2.30° C.) Y92P + F208W116% 86.60° C. +/− 0.17° C. (+1.90° C.) F208W + V170I 128% 85.80° C. +/−0.00° C. (+1.10° C.) F208W + D203C + 123% 94.80° C. +/− 0.00° C.(+10.10° C.) S248C F208I + D203C + 133% 90.90° C. +/− 0.00° C. (+6.20°C.) S248C

1. (canceled)
 2. An esterase variant which (i) has a polyester degrading activity, (ii) has at least 75% identity to the full length amino acid sequence set forth in SEQ ID NO: 1, and (iii) has at least one amino acid modification as compared to SEQ ID NO: 1, wherein the position of the amino acid modification is numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1 and one of said at least one amino acid modifications is at position D203.
 3. The esterase variant of claim 2, wherein the amino acid modification at position D203 is D203E, D203K, or D203R.
 4. The esterase variant of claim 2, which further comprises: a) at least one amino acid substitution at position S248; or b) further comprises at least one amino acid substitution at position E173.
 5. The esterase variant of claim 2, which exhibits increased thermostability as compared to the esterase of SEQ ID NO:
 1. 6. The esterase variant of claim 2, which has at least the amino acid substitution selected from D203E/K/R and exhibits increased thermostability as compared to the esterase of SEQ ID NO:
 1. 7. A nucleic acid encoding an esterase as defined in claim
 2. 8. An expression cassette or vector comprising the nucleic acid of claim
 7. 9. A host cell comprising the nucleic acid of claim
 7. 10. A method of producing an esterase comprising: (a) culturing the host cell according to claim 9 under conditions suitable to express the nucleic acid encoding the esterase; and (b) recovering said esterase from the cell culture.
 11. A composition comprising an esterase according to claim 2 and one or several excipients or additives.
 12. A method of degrading a plastic product containing at least one polyester comprising (a) contacting the plastic product with an esterase according to claim 2, thereby degrading the plastic product.
 13. The method of claim 12, further comprising (b) recovering monomers and/or oligomers resulting from the degradation of the at least one polyester.
 14. The method of claim 12, wherein the plastic product comprises at least one polyester selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylen terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), Polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends/mixtures of these materials.
 15. A polyester containing material comprising an esterase variant according to claim 2 and/or a host cell expressing said esterase variant and at least one polyester selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylen terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), Polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends/mixtures of these materials.
 16. A plastic compound comprising at least one polyester selected from polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylen terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), Polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene naphthalate (PEN) and blends/mixtures of these materials and an esterase variant according to claim 2 and/or a host cell expressing said esterase variant.
 17. A nucleic acid encoding an esterase as defined in claim
 2. 18. An expression cassette or vector comprising the nucleic acid of claim
 2. 19. A host cell comprising the nucleic acid of claim
 2. 20. A method of degrading a plastic product containing at least one polyester selected from polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT), comprising (a) contacting the plastic product with an esterase according to claim 2, thereby degrading the plastic product.
 21. A polyester containing material comprising at least one polyester selected from polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT), and an esterase variant according to claim 2 and/or a host cell expressing said esterase variant. 